Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in
Haibiao Gong1, Ilona Holcomb1, Aik Ooi1
1Fluidigm Corporation , South San Francisco, California 94080, United States.
Bioconjugate Chemistry
|December 23, 2015
Summary
This study presents a new method for multiplex protein detection by conjugating oligonucleotides to antibodies using click chemistry. This technique converts protein levels into detectable oligonucleotide signals, enabling sensitive and cost-effective proteomics research.
Area of Science:
- Biochemistry
- Molecular Biology
- Genomics
Background:
- Multiplex protein detection remains a challenge, limiting the application of high-throughput genomics tools in proteomics.
- Bridging genomics and proteomics requires methods to convert protein abundance into nucleic acid signals.
Purpose of the Study:
- To develop a simple and cost-effective method for preparing oligonucleotide-conjugated antibodies for multiplex protein detection.
- To establish an oligonucleotide extension reaction (OER) assay for sensitive protein quantification.
Main Methods:
- Antibodies were conjugated with azide-modified oligonucleotides using strain-promoted alkyne-azide cycloaddition (Cu-free click reaction).
- The OER assay utilizes pairs of antibody binders, each with a unique oligonucleotide, to generate a DNA template for qPCR detection.
- Optimization of reaction conditions and purification ensured high yield and assay performance.
Main Results:
- The click reaction-based conjugation is efficient and requires minimal hands-on time.
- The OER assay demonstrated high sensitivity, with dynamic ranges of 5-6 logs for specific protein targets (CSTB, MET, EpCAM, CASP3).
- Multiplexed detection of proteins in cell lysates and single cells was successfully achieved.
Conclusions:
- This novel antibody conjugation and OER assay provide an affordable platform for multiplex protein detection.
- The method has the potential to significantly accelerate proteomics research by enabling scalable and sensitive protein analysis.
